NER: Biological Nanomachines: Assembly and Function of Protein-DNA Nanostructures at the Single-Molecule Level

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Through evolution, biology has produced a remarkably diverse and efficient collection of proteins. These proteins assemble into a seemingly infinite variety of nanobiostructures, and they promote an extensive repertoire of chemical processes; hence, they comprise an interesting collection of biomolecular nanomachines. The proteins that are responsible for the maintenance and manipulation of DNA are one important subset of this collection. Their capacity to assemble with, and to alter the structure of, DNA is essential for all biological function. These proteins function in the packaging of DNA, the high-fidelity copying of genetic information, the reading of the genetic code and its conversion into RNA, the generation of genetic diversity, and the preservation of genetic and structural integrity of the genome. This project describes a new experimental approach to study the assembly and function of several of the nanomachines that function in these biological processes. The method involves the direct visualization by fluorescence microscopy, in real-time, of the assembly, disassembly, and movement of these nanobiostructures on single, optically-trapped DNA molecules using a novel, multi-port, laminar-flow, micro flow cell. This instrument will allows one to readily introduce an individual, optically-trapped DNA molecule sequentially into a series of reaction conditions, and to visualize the changes in structure/assembly of the molecules in real-time using multi-wavelength fluorescence microscopy. The successful development of this instrument will reveal important information about the structure and function of DNA-protein interactions that cannot be obtained using large ensembles of DNA molecules (where such information is often lost by averaging, or obscured by competing intermolecular interactions). This research will provide revolutionary new information about how proteins function to alter the structure of DNA. Furthermore, the experimental techniques can be applied to the study of many other nanoscale biostructures.